Regeneration of complex bone defects remains a significant clinical challenge. Multi-tool biofabrication has permitted the combination of various biomaterials to create multifaceted composites with tailorable mechanical properties and spatially controlled biological function. In this study we sought to use bioprinting to engineer nonviral gene activated constructs reinforced by polymeric micro-filaments. A gene activated bioink was developed using RGD-γ-irradiated alginate and nano-hydroxyapatite (nHA) complexed to plasmid DNA (pDNA). This ink was combined with bone marrow-derived mesenchymal stem cells (MSCs) and then co-printed with a polycaprolactone supporting mesh to provide mechanical stability to the construct. Reporter genes were first used to demonstrate successful cell transfection using this system, with sustained expression of the transgene detected over 14 days postbioprinting. Delivery of a combination of therapeutic genes encoding for bone morphogenic protein and transforming growth factor promoted robust osteogenesis of encapsulated MSCs in vitro, with enhanced levels of matrix deposition and mineralization observed following the incorporation of therapeutic pDNA. Gene activated MSC-laden constructs were then implanted subcutaneously, directly postfabrication, and were found to support superior levels of vascularization and mineralization compared to cell-free controls. These results validate the use of a gene activated bioink to impart biological functionality to three-dimensional bioprinted constructs.
Get full access to this article
View all access options for this article.
References
1.
EvansC.Using genes to facilitate the endogenous repair and regeneration of orthopaedic tissues. Int Orthop, 38, 1761, 2014.
2.
LiS.D., and HuangL.Non-viral is superior to viral gene delivery. J Control Release, 123, 181, 2007.
3.
SantosJ.L., PanditaD., RodriguesJ., PegoA.P., GranjaP.L., and TomasH.Non-viral gene delivery to mesenchymal stem cells: methods, strategies and application in bone tissue engineering and regeneration. Curr Gene Ther, 11, 46, 2011.
4.
OltonD., LiJ., WilsonM.E., RogersT., CloseJ., HuangL., KumtaP.N., and SfeirC.Nanostructured calcium phosphates (NanoCaPs) for non-viral gene delivery: influence of the synthesis parameters on transfection efficiency. Biomaterials, 28, 1267, 2007.
5.
XieY., ChenY., SunM., and PingQ.A mini review of biodegradable calcium phosphate nanoparticles for gene delivery. Curr Pharm Biotechnol, 14, 918, 2013.
6.
DangP.N., DwivediN., PhillipsL.M., YuX., HerbergS., BowermanC., SolorioL.D., MurphyW.L., and AlsbergE.Controlled dual growth factor delivery from microparticles incorporated within human bone marrow-derived mesenchymal stem cell aggregates for enhanced bone tissue engineering via endochondral ossification. Stem Cells Transl Med, 5, 206, 2016.
7.
LinY., TangW., WuL., JingW., LiX., WuY., LiuL., LongJ., and TianW.Bone regeneration by BMP-2 enhanced adipose stem cells loading on alginate gel. Histochem Cell Biol, 129, 203, 2008.
8.
CurtinC.M., CunniffeG.M., LyonsF.G., BesshoK., DicksonG.R., DuffyG.P., and O'BrienF.J.Innovative collagen nano-hydroxyapatite scaffolds offer a highly efficient non-viral gene delivery platform for stem cell-mediated bone formation. Adv Mater, 24, 749, 2012.
9.
ChoiS., YuX., JongpaiboonkitL., HollisterS.J., and MurphyW.L.Inorganic coatings for optimized non-viral transfection of stem cells. Sci Rep, 3, 1567, 2013.
10.
Gonzalez-FernandezT., TierneyE.G., CunniffeG.M., O'BrienF.J., and KellyD.J. Gene delivery of TGF-beta3 and BMP2 in an MSC-laden alginate hydrogel for articular cartilage and endochondral bone tissue engineering. Tissue Eng Part A, 22, 776, 2016.
11.
DoA.V., KhorsandB., GearyS.M., and SalemA.K.3D printing of scaffolds for tissue regeneration applications. Adv Healthc Mater, 4, 1742, 2015.
12.
MelchelsF.P., BlokzijlM.M., LevatoR., PeifferQ.C., RuijterM., HenninkW.E., VermondenT., and MaldaJ.Hydrogel-based reinforcement of 3D bioprinted constructs. Biofabrication, 8, 035004, 2016.
13.
MurphyS.V., and AtalaA.3D bioprinting of tissues and organs. Nat Biotechnol, 32, 773, 2014.
14.
VisserJ., MelchelsF.P.W., JeonJ.E., van BusselE.M., KimptonL.S., ByrneH.M., DhertW.J.A., DaltonP.D., HutmacherD.W., and MaldaJ.Reinforcement of hydrogels using three-dimensionally printed microfibres. Nat Commun, 6, 6933, 2015.
15.
MaldaJ., VisserJ., MelchelsF.P., JüngstT., HenninkW.E., DhertW.J.A., GrollJ., and HutmacherD.W.25th Anniversary Article: engineering Hydrogels for Biofabrication. Adv Mater, 25, 5011, 2013.
16.
CooperG.M., MillerE.D., DecesareG.E., UsasA., LensieE.L., BykowskiM.R., HuardJ., WeissL.E., LoseeJ.E., and CampbellP.G.Inkjet-based biopatterning of bone morphogenetic protein-2 to spatially control calvarial bone formation. Tissue Eng Part A, 16, 1749, 2010.
17.
BonadioJ., SmileyE., PatilP., and GoldsteinS.Localized, direct plasmid gene delivery in vivo: prolonged therapy results in reproducible tissue regeneration. Nat Med, 5, 753, 1999.
18.
LoozenL.D., WegmanF., OnerF.C., DhertW.J.A., and AlblasJ.Porous bioprinted constructs in BMP-2 non-viral gene therapy for bone tissue engineering. J Mater Chem B, 1, 6619, 2013.
19.
KrebsM.D., SalterE., ChenE., SutterK.A., and AlsbergE.Calcium phosphate-DNA nanoparticle gene delivery from alginate hydrogels induces in vivo osteogenesis. J Biomed Mater Res A, 92, 1131, 2010.
20.
BillietT., VandenhauteM., SchelfhoutJ., Van VlierbergheS., and DubruelP.A review of trends and limitations in hydrogel-rapid prototyping for tissue engineering. Biomaterials, 33, 6020, 2012.
21.
BoereK.W., VisserJ., SeyednejadH., RahimianS., GawlittaD., van SteenbergenM.J., DhertW.J., HenninkW.E., VermondenT., and MaldaJ.Covalent attachment of a three-dimensionally printed thermoplast to a gelatin hydrogel for mechanically enhanced cartilage constructs. Acta Biomater, 10, 2602, 2014.
22.
XuT., BinderK.W., AlbannaM.Z., DiceD., ZhaoW., YooJ.J., and AtalaA.Hybrid printing of mechanically and biologically improved constructs for cartilage tissue engineering applications. Biofabrication, 5, 015001, 2013.
23.
DalyA.C., CunniffeG.M., SathyB.N., JeonO., AlsbergE., and KellyD.J.3D Bioprinting of Developmentally Inspired Templates for Whole Bone Organ Engineering. Adv Healthc Mater, 5, 2353, 2016.
24.
SchuurmanW., LevettP.A., PotM.W., van WeerenP.R., DhertW.J.A., HutmacherD.W., MelchelsF.P.W., KleinT.J., and MaldaJ.Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs. Macromol Biosci, 13, 551, 2013.
25.
CunniffeG.M., O'BrienF.J., PartapS., LevingstoneT.J., StantonK.T., and DicksonG.R.The synthesis and characterization of nanophase hydroxyapatite using a novel dispersant-aided precipitation method. J Biomed Mater Res A, 95, 1142, 2010.
26.
AlsbergE., KongH.J., HiranoY., SmithM.K., AlbeirutiA., and MooneyD.J.Regulating bone formation via controlled scaffold degradation. J Dent Res, 82, 903, 2003.
27.
JeonO., PowellC., AhmedS.M., and AlsbergE.Biodegradable, photocrosslinked alginate hydrogels with independently tailorable physical properties and cell adhesivity. Tissue Eng Part A, 16, 2915, 2010.
28.
Ignat'evaN.Y., DanilovN.A., AverkievS.V., ObrezkovaM.V., LuninV.V., and SobolE.N.Determination of hydroxyproline in tissues and the evaluation of the collagen content of the tissues. J Anal Chem, 62, 51, 2007.
29.
CunniffeG.M., VinardellT., MurphyJ.M., ThompsonE.M., MatsikoA., O'BrienF.J., and KellyD.J.Porous decellularized tissue engineered hypertrophic cartilage as a scaffold for large bone defect healing. Acta Biomater, 23, 82, 2015.
30.
SimmonsC.A., AlsbergE., HsiongS., KimW.J., and MooneyD.J.Dual growth factor delivery and controlled scaffold degradation enhance in vivo bone formation by transplanted bone marrow stromal cells. Bone, 35, 562, 2004.
31.
CunniffeG.M., VinardellT., ThompsonE.M., DalyA.C., MatsikoA., O'BrienF.J., and KellyD.J.Chondrogenically primed mesenchymal stem cell-seeded alginate hydrogels promote early bone formation in critically-sized defects. Eur Polym J, 72, 464, 2015.
32.
KunduJ., ShimJ.H., JangJ., KimS.W., and ChoD.W.An additive manufacturing-based PCL–alginate–chondrocyte bioprinted scaffold for cartilage tissue engineering. J Tissue Eng Regen Med, 9, 1286, 2015.
33.
VenkatesanJ., BhatnagarI., ManivasaganP., KangK.H., and KimS.K.Alginate composites for bone tissue engineering: a review. Int J Biol Macromol, 72, 269, 2015.
34.
WegmanF., BijenhofA., SchuijffL., OnerF.C., DhertW.J., and AlblasJ.Osteogenic differentiation as a result of BMP-2 plasmid DNA based gene therapy in vitro and in vivo. Eur Cell Mater, 21, 230, 2011.
35.
SadrN., PippengerB.E., ScherberichA., WendtD., ManteroS., MartinI., and PapadimitropoulosA.Enhancing the biological performance of synthetic polymeric materials by decoration with engineered, decellularized extracellular matrix. Biomaterials, 33, 5085, 2012.
36.
PatiF., SongT.H., RijalG., JangJ., KimS.W., and ChoD.W.Ornamenting 3D printed scaffolds with cell-laid extracellular matrix for bone tissue regeneration. Biomaterials, 37, 230, 2015.
37.
ShimJ.H., KimS.E., ParkJ.Y., KunduJ., KimS.W., KangS.S., and ChoD.W.Three-dimensional printing of rhBMP-2-loaded scaffolds with long-term delivery for enhanced bone regeneration in a rabbit diaphyseal defect. Tissue Eng Part A, 20, 1980, 2014.
38.
LeeH.J., KimY.B., AhnS.H., LeeJ.S., JangC.H., YoonH., ChunW., and KimG.H.A new approach for fabricating collagen/ECM-based bioinks using preosteoblasts and human adipose stem cells. Adv Healthc Mater, 4, 1359, 2015.
39.
KimY.B., and KimG.H.PCL/Alginate Composite Scaffolds for Hard Tissue Engineering: fabrication, Characterization, and Cellular Activities. ACS Comb Sci, 17, 87, 2015.
40.
GrandiC., Di LiddoR., PaganinP., LoraS., DalzoppoD., FeltrinG., GiraudoC., TommasiniM., ConconiM.T., and ParnigottoP.P.Porous alginate/poly(epsilon-caprolactone) scaffolds: preparation, characterization and in vitro biological activity. Int J Mol Med, 27, 455, 2011.
41.
WegmanF., van der HelmY., OnerF.C., DhertW.J., and AlblasJ.Bone morphogenetic protein-2 plasmid DNA as a substitute for bone morphogenetic protein-2 protein in bone tissue engineering. Tissue Eng Part A, 19, 2686, 2013.
42.
AugstA.D., KongH.J., and MooneyD.J.Alginate Hydrogels as Biomaterials. Macromol Biosci, 6, 623, 2006.
43.
SheehyE.J., MesallatiT., VinardellT., and KellyD.J.Engineering cartilage or endochondral bone: a comparison of different naturally derived hydrogels. Acta Biomater, 13, 245, 2015.
44.
LoozenL.D., van der HelmY.J.M., ÖnerF.C., DhertW.J.A., KruytM.C., and AlblasJ.Bone Morphogenetic Protein-2 Nonviral Gene Therapy in a Goat Iliac Crest Model for Bone Formation. Tissue Eng Part A, 21, 1672, 2015.
45.
CurtinC.M., TierneyE.G., McSorleyK., CryanS.A., DuffyG.P., and O'BrienF.J.Combinatorial gene therapy accelerates bone regeneration: non-viral dual delivery of VEGF and BMP2 in a collagen-nanohydroxyapatite scaffold. Adv Healthc Mater, 4, 223, 2014.
46.
CunniffeG.M., DicksonG.R., PartapS., StantonK.T., and O'BrienF.J.Development and characterisation of a collagen nano-hydroxyapatite composite scaffold for bone tissue engineering. J Mater Sci Mater Med, 21, 2293, 2010.
47.
CunniffeG.M., CurtinC.M., ThompsonE.M., DicksonG.R., and O'BrienF.J.Content-dependent osteogenic response of nanohydroxyapatite: an in vitro and in vivo assessment within collagen-based scaffolds. ACS Appl Mater Interfaces, 8, 23477, 2016.
48.
OestM.E., DupontK.M., KongH.J., MooneyD.J., and GuldbergR.E.Quantitative assessment of scaffold and growth factor-mediated repair of critically sized bone defects. J Orthop Res, 25, 941, 2007.
Supplementary Material
Please find the following supplemental material available below.
For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.
For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.
